Summary of Work: The goal of this project is to understand the process of eukaryotic post-replication mismatch repair, which selectively removes DNA replication errors from the newly synthesized strand. We are examining the ability of normal and mutant human cell extracts to correct DNA substrates containing mispaired or unpaired nucleotides. Mismatch repair activity is defective in cancer cell lines contaning mutations in any of five different genes, MSH2, MSH3, MSH6, MLH1, and PMS2. Each of these lines has been corrected by transfer of a wild-type chromosome to the cell line and the resulting phenotypes are being examined. These phenotypes include microsatellite instability, HPRT mutation rate, sensitivity to base analogues and alkylating agents, cell cycle checkpoint control, and mismatch repair, at a step preceding DNA resynthesis. We have demonstrated that MLH1 is required for mismatch repoari by studying the repair capacity of fibroblast cells derived from mice that are (+/+), (+/-) and (-/-) for the MLH1 gene. We have demonstrated that fibroblast cells derived from Werner Syndrome, a premature aging syndrome, are defective in mismatch repair. These studies are important for understanding the genetics and biochemistry of the mismatch repair system, the multiple functions of the numerous mismatch repair genes, the molecular genetic basis for the initiating events in cancer and its subsequent treatment and the risk posed to individuals in the population by exposure to DNA damaging agents.